Our goal is to develop a viral vector to study the molecular interactions between arthropods and the pathogens they transmit causing diseases such as malaria, lymphatic filariasis, dengue fever, African sleeping sickness, Chaga's disease and Lyme disease. A viral vector for gene expression and gene silencing in arthropods is a powerful tool for studying the function of the genes involved in disease transmission. Vectors derived from Sindbis virus (SIN) have proven to be very useful in this type of research and few other viral vectors are under development. Use of SIN, however, is constrained by the narrow host range, limited tissue dissemination, inability to express larger genes and human pathogenicity of the virus. During the previous period of support, we initiated studies to develop a highly efficient viral vector system based on Flock House Virus (FHV), a simple RNA virus and a member of the Nodaviridae family of insect origin. FHV has a small genome (4.5 kb) composed of two separate positive sense single stranded RNAs packaged into a coat protein with no envelope. It is not a human pathogen and has the ability to adapt to a wide range of insect hosts as well as hosts from different kingdoms such as plants and yeast. During the initial period of support we have extended the host range of FHV to four mosquito genera (Aedes, Anopheles, Culex and Armigeres), a species of tsetse (Glossina morsitans) and a reduviid (Rhodnius prolixus). These insects showed no adverse effect when infected with appropriate doses of FHV. We have made several vector constructs and demonstrated their abilities to express forein genes in mosquito and tsetse cells. In this continuation application we propose to (1) assess infection potential of FHV to two species of ticks (Ixodes scapularis and Ornithodoros turacata) that transmit Lyme disease and Relapsing Fever (2) continue to improve existing FHV RNA-based vectors for transient gene expression in above insect cells, and develop DNA-based vectors for prolonged expression, and (3) use FHV vectors to study the effects of expressing and/or silencing genes involved in immunity and pathogen transmissibility of Anopheles, tsetse and ticks. We expect to establish FHV vectors as a powerful new system to study gene function in a wide variety of medically important arthropods.